Combining adoptive NK cell infusion with a dopamine-releasing peptide reduces senescent cells in aged mice

Aging inducing the development of senescent cells (SNCs) in various tissues is considered as the main cause of the age-related diseases. Senotherapy has become a promising anti-aging therapy. However, the effectivity and side-effect of senolytic agents are still concern. Here, we observed the downregulation of senescence-related genes by adoptive infusion of natural killer (NK) cells in 26 cases in peripheral blood CD3⁺ T cells. NK cell treatment also significantly decreased levels of senescence markers and senescence-associated secretory phenotypes (SASPs) in three senescent adipose tissues when culturing them together. Interestingly, cytotoxic activity of mouse NK cells against SNCs was significantly enhanced by dopamine in vitro through D1-like receptors. Acein, dopamine-releasing peptide, promoted the adoptive infusion of NK cells in effectively eliminating SNCs in a variety of tissues and reduced local and systemic SASPs in aging mice but Acein alone did not have the senolytic effect. These data demonstrated that adoptive infusion of NK cells is an effective means in removing SNCs, and peptide Acein combined with NK cells further enhances this effect in aging mice.Subject terms: Immunotherapy, Diseases     

Identification of a novel senolytic agent,navitoclax, targeting the Bcl‐2 family of anti‐apoptotic factors

Clearing senescent cells extends healthspan in mice. Using a hypothesis‐driven bioinformatics‐based approach, we recently identified pro‐survival pathways in human senescent cells that contribute to their resistance to apoptosis. This led to identification of dasatinib (D) and quercetin (Q) as senolytics, agents that target some of these pathways and induce apoptosis preferentially in senescent cells. Among other pro‐survival regulators identified was Bcl‐xl. Here, we tested whether the Bcl‐2 family inhibitors, navitoclax (N) and TW‐37 (T), are senolytic. Like D and Q, N is senolytic in some, but not all types of senescent cells: N reduced viability of senescent human umbilical vein epithelial cells (HUVECs), IMR90 human lung fibroblasts, and murine embryonic fibroblasts (MEFs), but not human primary preadipocytes, consistent with our previous finding that Bcl‐xl siRNA is senolytic in HUVECs, but not preadipocytes. In contrast, T had little senolytic activity. N targets Bcl‐2, Bcl‐xl, and Bcl‐w, while T targets Bcl‐2, Bcl‐xl, and Mcl‐1. The combination of Bcl‐2, Bcl‐xl, and Bcl‐w siRNAs was senolytic in HUVECs and IMR90 cells, while combination of Bcl‐2, Bcl‐xl, and Mcl‐1 siRNAs was not. Susceptibility to N correlated with patterns of Bcl‐2 family member proteins in different types of human senescent cells, as has been found in predicting response of cancers to N. Thus, N is senolytic and acts in a potentially predictable cell type‐restricted manner. The hypothesis‐driven, bioinformatics‐based approach we used to discover that dasatinib (D) and quercetin (Q) are senolytic can be extended to increase the repertoire of senolytic drugs, including additional cell type‐specific senolytic agents.     

Preclinical studies for improving radiosensitivity of non-small cell lung cancer cell lines by combining glutaminase inhibition and senolysis

Glutamine metabolism, known as glutaminolysis, is abnormally activated in many cancer cells with KRAS or BRAF mutations or active c-MYC. Glutaminolysis plays an important role in the proliferation of cancer cells with oncogenic mutations. In this study, we characterized radiation-induced cell death, which was enhanced by glutaminolysis inhibition in non-small cell lung cancer A549 and H460 cell lines with KRAS mutation. A clonogenic survival assay revealed that treatment with a glutaminase inhibitor, CB839, enhanced radiosensitivity. X-irradiation increased glutamate production, mitochondrial oxygen consumption, and ATP production, whereas CB839 treatment suppressed these effects. The data suggest that the enhancement of glutaminolysis-dependent energy metabolism for ATP production is important for survival after X-irradiation. Evaluation of the cell death phenotype revealed that glutaminolysis inhibitory treatment with CB839 or a low-glutamine medium significantly promoted the proliferation of β-galactosidase-positive and IL-6/IL-8 secretory cells among X-irradiated tumor cells, corresponding to an increase in the senescent cell population. Furthermore, treatment with ABT263, a Bcl-2 family inhibitor, transformed senescent cells into apoptotic cells. The findings suggest that combination treatment with a glutaminolysis inhibitor and a senolytic drug is useful for efficient radiotherapy.     

Cellular senescence in the aging and diseased kidney

The program of cellular senescence is involved in both the G1 and G2 phase of the cell cycle, limiting G1/S and G2/M progression respectively, and resulting in prolonged cell cycle arrest. Cellular senescence is involved in normal wound healing. However, multiple organs display increased senescent cell numbers both during natural aging and after injury, suggesting that senescent cells can have beneficial as well as detrimental effects in organismal aging and disease. Also in the kidney, senescent cells accumulate in various compartments with advancing age and renal disease. In experimental studies, forced apoptosis induction through the clearance of senescent cells leads to better preservation of kidney function during aging. Recent groundbreaking studies demonstrate that senescent cell depletion through INK-ATTAC transgene-mediated or cell-penetrating FOXO4-DRI peptide induced forced apoptosis, reduced age-associated damage and dysfunction in multiple organs, in particular the kidney, and increased performance and lifespan. Senescence is also involved in oncology and therapeutic depletion of senescent cells by senolytic drugs has been studied in experimental and human cancers. Although studies with senolytic drugs in models of kidney injury are lacking, their dose limiting side effects on other organs suggest that targeted delivery might be needed for successful application of senolytic drugs for treatment of kidney disease. In this review, we discuss (i) current understanding of the mechanisms and associated pathways of senescence, (ii) evidence of senescence occurrence and causality with organ injury, and (iii) therapeutic strategies for senescence depletion (senotherapy) including targeting, all in the context of renal aging and disease.     

A rationally designed fluorescence probe achieves highly specific and long-term detection of senescence in vitro and in vivo

Senescent cells (SnCs) are implicated in aging and various age-related pathologies. Targeting SnCs can treat age-related diseases and extend health span. However, precisely tracking and visualizing of SnCs is still challenging, especially in in vivo environments. Here, we developed a near-infrared (NIR) fluorescent probe (XZ1208) that targets β-galactosidase (β-Gal), a well-accepted biomarker for cellular senescence. XZ1208 can be cleaved rapidly by β-Gal and produces a strong fluorescence signal in SnCs. We demonstrated the high specificity and sensitivity of XZ1208 in labeling SnCs in naturally aged, total body irradiated (TBI), and progeroid mouse models. XZ1208 achieved a long-term duration of over 6 days in labeling senescence without causing significant toxicities and accurately detected the senolytic effects of ABT263 on eliminating SnCs. Furthermore, XZ1208 was applied to monitor SnCs accumulated in fibrotic diseases and skin wound healing models. Overall, we developed a tissue-infiltrating NIR probe and demonstrated its excellent performance in labeling SnCs in aging and senescence-associated disease models, indicating great potential for application in aging studies and diagnosis of senescence-associated diseases.     

Senolytic activity of piperlongumine analogues: Synthesis and biological evaluation

Selective clearance of senescent cells (SCs) has emerged as a potential therapeutic approach for age-related diseases, as well as chemotherapy- and radiotherapy-induced adverse effects. Through a cell-based phenotypic screening approach, we recently identified piperlongumine (PL), a dietary natural product, as a novel senolytic agent, referring to small molecules that can selectively kill SCs over normal or non-senescent cells. In an effort to establish the structure-senolytic activity relationships of PL analogues, we performed a series of structural modifications on the trimethoxyphenyl and the α,β-unsaturated δ-valerolactam rings of PL. We show that modifications on the trimethoxyphenyl ring are well tolerated, while the Michael acceptor on the lactam ring is critical for the senolytic activity. Replacing the endocyclic C2–C3 olefin with an exocyclic methylene at C2 render PL analogues 47–49 with increased senolytic activity. These α-methylene containing analogues are also more potent than PL in inducing ROS production in WI-38 SCs. Similar to PL, 47–49 reduce the protein levels of oxidation resistance 1 (OXR1), an important oxidative stress response protein that regulates the expression of a variety of antioxidant enzymes, in cells. This study represents a useful starting point toward the discovery of senolytic agents for therapeutic uses.     

Galacto‐conjugation of Navitoclax as an efficient strategy to increase senolytic specificity and reduce platelet toxicity

Pharmacologically active compounds with preferential cytotoxic activity for senescent cells, known as senolytics, can ameliorate or even revert pathological manifestations of senescence in numerous preclinical mouse disease models, including cancer models. However, translation of senolytic therapies to human disease is hampered by their suboptimal specificity for senescent cells and important toxicities that narrow their therapeutic windows. We have previously shown that the high levels of senescence‐associated lysosomal β‐galactosidase (SA‐β‐gal) found within senescent cells can be exploited to specifically release tracers and cytotoxic cargoes from galactose‐encapsulated nanoparticles within these cells. Here, we show that galacto‐conjugation of the BCL‐2 family inhibitor Navitoclax results in a potent senolytic prodrug (Nav‐Gal), that can be preferentially activated by SA‐β‐gal activity in a wide range of cell types. Nav‐Gal selectively induces senescent cell apoptosis and has a higher senolytic index than Navitoclax (through reduced activation in nonsenescent cells). Nav‐Gal enhances the cytotoxicity of standard senescence‐inducing chemotherapy (cisplatin) in human A549 lung cancer cells. Concomitant treatment with cisplatin and Nav‐Gal in vivo results in the eradication of senescent lung cancer cells and significantly reduces tumour growth. Importantly, galacto‐conjugation reduces Navitoclax‐induced platelet apoptosis in human and murine blood samples treated ex vivo, and thrombocytopenia at therapeutically effective concentrations in murine lung cancer models. Taken together, we provide a potentially versatile strategy for generating effective senolytic prodrugs with reduced toxicities.     

Aging-associated increase of gelsolin for apoptosis resistance

Gelsolin, a Ca(2+)-dependent actin regulatory protein, was recently suggested to participate in apoptosis regulation. In this study, we found that the level of gelsolin is elevated in senescent human diploid fibroblasts (HDFs) and also in the tissues of old rats, i.e., in the liver, kidney, heart, spleen, stomach, and brain, etc. The ubiquitous increase of gelsolin in the aged organs and cells led us to assume that it might be related with one of the cardinal senescent phenotypes, aging-associated apoptosis resistency. Thus, we tested the sensitivity of senescent cells to apoptosis by menadione, an apoptosis-inducing agent, before and after the down-regulation of gelsolin. The down-regulation of gelsolin in senescent HDFs, independently of Bcl-2 family expression, resulted in an increased sensitivity to menadione-induced apoptotic cell death. The observed ubiquitous increase of gelsolin in the senescent states of cells and tissues, and the increased sensitivity to apoptosis-induction by gelsolin down-regulation, suggests that gelsolin would be partly responsible for age-related apoptosis resistance.     

Protective roles of cytoplasmic p21Cip1 /Waf1 in senolysis and ferroptosis of lung cancer cells

ObjectiveTherapy‐induced senescent cancer cells increase the expression of the cyclin‐dependent kinase inhibitors p16^Ink4a and p21^Cip1/Waf1. Given that p21 regulates not only the cell cycle but also cell death, we investigated the roles of p21 in cell death using a p16‐negative A549 human lung adenocarcinoma cell line.MethodsSenescence was induced by doxorubicin (DXR) or pemetrexed (PEM). The protein expression of p21 was examined by immunoblot. Cell death, reactive oxygen species (ROS) and lipid peroxidation were determined by flow cytometry. ABT‐263 and ABT‐737 were used as senolytic drugs. In vivo growth of A549 cells with different levels of p21 and their sensitivity to PEM were examined in xenograft models.ResultsDXR‐induced senescent A549 cells increased the expression of cytoplasmic p21, and the sensitivity to ABT‐263 was augmented in p21‐knockout A549 (A549‐KOp21) cells. A similar senolytic effect was observed when PEM was combined with ABT‐737. PEM alone induced a higher level of non‐apoptotic cell death, ferroptosis, in A549‐KOp21 cells than in A549 cells. Although there was no difference in the level of lipid peroxidation, ROS levels were higher in PEM‐treated A549‐KOp21 cells than in PEM‐treated A549 cells. A loss of p21 increased the sensitivity of A549 cells to PEM both in vitro and in vivo. A clinical database analysis showed that CDKN1A ^high lung adenocarcinoma patients had a poorer prognosis compared to CDKN1A ^low patients.ConclusionCytoplasmic p21, which was increased in therapy‐induced senescent lung cancer cells, plays protective roles in senolysis and ferroptosis.

Doxorubicin or pemetrexed could induce senescence in p21‐expressing parental A549 cells and increase the expression of cytoplasmic p21. However, such drug‐induced senescent A549 cells were relatively resistant to apoptosis by senolytic drugs. By contrast, p21‐knockout A549 (A549‐KOp21) cells increased their sensitivity to senolytic drugs. On the other hand, pemetrexed induced a higher level of non‐apoptotic cell death, ferroptosis, in A549‐KOp21 cells than in A549 cells. These findings highlight the protective roles of cytoplasmic p21 against senolysis and ferroptosis in therapy‐induced senescent lung cancer cells.     

Is exercise a senolytic medicine? A systematic review

Cellular senescence, a state of irreversible growth arrest triggered by various stressors, engages in a category of pathological processes, whereby senescent cells accumulate in mitotic tissues. Senolytics as novel medicine against aging and various diseases through the elimination of senescent cells has emerged rapidly in recent years. Exercise is a potent anti‐aging and anti‐chronic disease medicine, which has shown the capacity to lower the markers of cellular senescence over the past decade. However, whether exercise is a senolytic medicine for aging and various diseases remains unclear. Here, we have conducted a systematic review of the published literature studying the senolytic effects of exercise or physical activity on senescent cells under various states in both human and animal models. Exercise can reduce the markers of senescent cells in healthy humans, while it lowered the markers of senescent cells in obese but not healthy animals. The discrepancy between human and animal studies may be due to the relatively small volume of research and the variations in markers of senescent cells, types of cells/tissues, and health conditions. These findings suggest that exercise has senolytic properties under certain conditions, which warrant further investigations.     

Regulation of Survival Networks in Senescent Cells: From Mechanisms to Interventions

Cellular senescence is a state of stable cell cycle arrest arising in response to DNA and mitochondrial damages. Senescent cells undergo morphological, structural and functional changes that are influenced by a number of variables, including time, stress, tissue, and cell type. The heterogeneity of the senescent phenotype is exemplified by the many biological properties that senescent cells can cover. The advent of innovative model organisms has demonstrated a functional role of senescent cells during embryogenesis, tissue remodeling, tumorigenesis and aging. Importantly, prolonged and aberrant persistence of senescent cells is often associated with tissue dysfunction and pathology, and is partially the consequence of mechanisms that enhance survival and resistance to cell death. Here, we describe the main molecular players involved in promoting survival of senescent cells, with particular emphasis on the regulation of senescence-associated anti-apoptotic pathways. We discuss the consequences these pathways have in providing resistance to intrinsic and extrinsic pro-apoptotic signals. Finally, we highlight the importance of these pathways in the development of targets for senolytic interventions.     

Oxidation resistance 1 is a novel senolytic target

The selective depletion of senescent cells (SCs) by small molecules, termed senolytic agents, is a promising therapeutic approach for treating age‐related diseases and chemotherapy‐ and radiotherapy‐induced side effects. Piperlongumine (PL) was recently identified as a novel senolytic agent. However, its mechanism of action and molecular targets in SCs was unknown and thus was investigated. Specifically, we used a PL‐based chemical probe to pull‐down PL‐binding proteins from live cells and then mass spectrometry‐based proteomic analysis to identify potential molecular targets of PL in SCs. One prominent target was oxidation resistance 1 (OXR1), an important antioxidant protein that regulates the expression of a variety of antioxidant enzymes. We found that OXR1 was upregulated in senescent human WI38 fibroblasts. PL bound to OXR1 directly and induced its degradation through the ubiquitin‐proteasome system in an SC‐specific manner. The knockdown of OXR1 expression by RNA interference significantly increased the production of reactive oxygen species in SCs in conjunction with the downregulation of antioxidant enzymes such as heme oxygenase 1, glutathione peroxidase 2, and catalase, but these effects were much less significant when OXR1 was knocked down in non‐SCs. More importantly, knocking down OXR1 selectively induced apoptosis in SCs and sensitized the cells to oxidative stress caused by hydrogen peroxide. These findings provide new insights into the mechanism by which SCs are highly resistant to oxidative stress and suggest that OXR1 is a novel senolytic target that can be further exploited for the development of new senolytic agents.     

Protein phosphatase 2A activators reverse age-related behavioral changes by targeting neural cell senescence

The contribution of cellular senescence to the behavioral changes observed in the elderly remains elusive. Here, we observed that aging is associated with a decline in protein phosphatase 2A (PP2A) activity in the brains of zebrafish and mice. Moreover, drugs activating PP2A reversed age-related behavioral changes. We developed a transgenic zebrafish model to decrease PP2A activity in the brain through knockout of the ppp2r2c gene encoding a regulatory subunit of PP2A. Mutant fish exhibited the behavioral phenotype observed in old animals and premature accumulation of neural cells positive for markers of cellular senescence, including senescence-associated β-galactosidase, elevated levels cdkn2a/b, cdkn1a, senescence-associated secretory phenotype gene expression, and an increased level of DNA damage signaling. The behavioral and cell senescence phenotypes were reversed in mutant fish through treatment with the senolytic ABT263 or diverse PP2A activators as well as through cdkn1a or tp53 gene ablation. Senomorphic function of PP2A activators was demonstrated in mouse primary neural cells with downregulated Ppp2r2c. We conclude that PP2A reduction leads to neural cell senescence thereby contributing to age-related behavioral changes and that PP2A activators have senotherapeutic properties against deleterious behavioral effects of brain aging.     

Rag1 immunodeficiency‐induced early aging and senescence in zebrafish are dependent on chronic inflammation and oxidative stress

In mammals, recombination activating gene 1 (RAG1) plays a crucial role in adaptive immunity, generating a vast range of immunoglobulins. Rag1^?/? zebrafish (Danio rerio) are viable and reach adulthood without obvious signs of infectious disease in standard nonsterile conditions, suggesting that innate immunity could be enhanced to compensate for the lack of adaptive immunity. By using microarray analysis, we confirmed that the expression of immunity‐ and apoptosis‐related genes was increased in the rag1^?/? fish. This tool also allows us to notice alterations of the DNA repair and cell cycle mechanisms in rag1^?/? zebrafish. Several senescence and aging markers were analyzed. In addition to the lower lifespan of rag1^?/? zebrafish compared to their wild‐type (wt) siblings, rag1^?/? showed a higher incidence of cell cycle arrest and apoptosis, a greater amount of phosphorylated histone H2AX, oxidative stress and decline of the antioxidant mechanisms, an upregulated expression and activity of senescence‐related genes and senescence‐associated β‐galactosidase, respectively, diminished telomere length, and abnormal self‐renewal and repair capacities in the retina and liver. Metabolomic analysis also demonstrated clear differences between wt and rag1^?/? fish, as was the deficiency of the antioxidant metabolite l ‐acetylcarnitine (ALCAR) in rag1^?/? fish. Therefore, Rag1 activity does not seem to be limited to V(D)J recombination but is also involved in senescence and aging. Furthermore, we confirmed the senolytic effect of ABT‐263, a known senolytic compound and, for the first time, the potential in vivo senolytic activity of the antioxidant agent ALCAR, suggesting that this metabolite is essential to avoid premature aging.     

Induction of apoptosis in human replicative senescent fibroblasts

Cellular senescence is an irreversible growth phase characteristic of normal cells. We have found that human senescent fibroblasts can be induced to undergo programmed cell death (apoptosis) by ceramide, TNF-alpha, or okadaic acid. The most profound effects were induced by TNF-alpha and okadaic acid treatment. In the present study, we also evaluated the contribution of lysosomal activation as a possible mechanism underlying the induction of apoptosis. Four lysosomal enzyme activities were measured: beta-galactosidase, alpha-galactosidase A, beta-glucoronidase, and acid phosphatase. Using an in situ assay, we have found that the activity of beta-galactosidase, which is also a biochemical marker of senescence, is induced in young proliferating fibroblasts following exposure to all three apoptotic inducing agents. The other enzymes were not significantly induced in young fibroblasts following exposure to agents that induce apoptosis. During replicative senescence, three of the four lysosomal enzymes tested (beta-galactosidase, alpha-galactosidase A, and beta-glucoronidase) are constitutively expressed at high levels. TNF-alpha was the only agent that induced lysosomal activity in senescent fibroblasts, of which only alpha-galactosidase A activity was induced. Our studies show that senescent fibroblasts can be induced to undergo apoptosis in a signal-dependent manner. However, the lysosomal enzymes examined do not appear to be correlated with apoptotic induction.     

Efficient elimination of cancer cells by deoxyglucose-ABT-263/737 combination therapy

As single agents, ABT-263 and ABT-737 (ABT), molecular antagonists of the Bcl-2 family, bind tightly to Bcl-2, Bcl-xL and Bcl-w, but not to Mcl-1, and induce apoptosis only in limited cell types. The compound 2-deoxyglucose (2DG), in contrast, partially blocks glycolysis, slowing cell growth but rarely causing cell death. Injected into an animal, 2DG accumulates predominantly in tumors but does not harm other tissues. However, when cells that were highly resistant to ABT were pre-treated with 2DG for 3 hours, ABT became a potent inducer of apoptosis, rapidly releasing cytochrome c from the mitochondria and activating caspases at submicromolar concentrations in a Bak/Bax-dependent manner. Bak is normally sequestered in complexes with Mcl-1 and Bcl-xL. 2DG primes cells by interfering with Bak-Mcl-1 association, making it easier for ABT to dissociate Bak from Bcl-xL, freeing Bak to induce apoptosis. A highly active glucose transporter and Bid, as an agent of the mitochondrial apoptotic signal amplification loop, are necessary for efficient apoptosis induction in this system. This combination treatment of cancer-bearing mice was very effective against tumor xenograft from hormone-independent highly metastasized chemo-resistant human prostate cancer cells, suggesting that the combination treatment may provide a safe and effective alternative to genotoxin-based cancer therapies.     

清除衰老细胞在衰老与老龄化相关疾病中的研究进展

衰老是一个新兴的重要研究领域,随着该领域相关知识的积累和技术的进步,人们逐渐意识到衰老本身可以被针对性地干预,实现延长寿命并且延缓衰老相关疾病的发生发展,具有重要的科学和现实意义.引起个体衰老的众多因素中,衰老细胞的积累被认为是导致器官衰老发生退行性变,最终引起衰老相关疾病的重要原因.近年来,多项研究表明,清除体内衰老细胞可以延缓多种衰老相关疾病的发生,直接证明了衰老细胞是导致衰老相关疾病的重要原因之一,为治疗衰老相关疾病提供了新靶点.细胞衰老是由于损伤积累诱发了细胞周期抑制通路的激活,细胞永久地退出细胞增殖周期.衰老细胞会发生细胞形态、转录谱、蛋白质稳态、表观遗传以及代谢等系列特征的改变,同时衰老细胞对凋亡发生抵抗从而在体内多器官组织积累.衰老细胞会激活炎症因子分泌通路,导致组织局部非感染性炎症微环境,进而导致器官退行性变及多种衰老相关疾病的发生发展.因此针对衰老细胞对凋亡抵抗的特性,多个研究小组通过筛选小分子化合物库,发现某些化合物能够选择性清除衰老细胞,这些小分子化合物被称为"senolytics",意为"衰老细胞杀伤性化合物".衰老细胞杀伤性化合物在多种衰老相关疾病动物模型中能够延缓疾病的发展并延长哺乳动物寿命.因此,靶向杀伤衰老细胞对多种衰老相关疾病的治疗从而提高健康寿命具有重要的临床应用前景.除靶向杀伤衰老细胞策略以外,干细胞移植、基因编辑、异体共生等策略在抗衰老研究发展中也具有重要意义,具有启发性.本文通过汇总近期衰老细胞清除领域的重要进展和多种抗衰老策略,将细胞衰老研究发展史做简要梳理,就细胞衰老与衰老相关疾病的关系作一综述,重点讨论衰老细胞在多种衰老相关疾病中作为治疗靶点的应用潜力,并就其局限性和进一步的研究方向进行探讨.